Energy beam guide for an electrophoresis system

ABSTRACT

The energy beam guide comprises a first region having a first refractive index, the first region having an energy beam receiving end and an inclined first boundary opposing the energy beam receiving end. The energy beam guide also includes a second region having a second refractive index that is less than the first refractive index. The second region shares the first boundary with the first region, and has a declined second boundary opposing the first boundary. A predetermined distance separates the first and second boundaries. Finally, the energy beam guide comprises a third region having a third refractive index. The third region shares the second boundary with the second region. Also provided are a method for making and using the energy beam guide.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. patent application Ser.No. 09/849,380, filed May 4, 2001, which is incorporated herein byreference.

TECHNICAL FIELD

[0002] The invention relates generally to an electrophoresis system.More particularly, the invention is directed to a detection cell forreceiving a sample to be analyzed photometrically, where the detectioncell acts as light guide for excitation light used to detect separatedchemical components.

BACKGROUND OF THE INVENTION

[0003] In biotechnology, separation and analysis of chemical samples iscritically important. Moreover, it is desirable to conduct multipleseparations and analyses of the separated components simultaneously toincrease the speed and efficiency at which chemical samples areevaluated. For example, separation technologies such as electrophoresisare used in DNA sequencing, protein molecular weight determination,genetic mapping, and other types of processes used to gather largeamounts of analytical information about particular chemical samples.

[0004] One method used to separate chemical samples into their componentparts is electrophoresis. Electrophoresis is the migration of chargedcolloidal particles or molecules through a solution under the influenceof an applied electric field usually provided by immersed electrodes,where the colloidal particles are a suspension of finely dividedparticles in a continuous medium.

[0005] Historically, a polymer gel containing the finely dividedparticles is placed between two glass plates and an electric fieldapplied to both ends of the plates. This method, however, offers a lowlevel of automation and long analysis times. More recently, thecapillary electrophoresis (hereafter “CE”) method was developed, whichhas the added advantages of speed, versatility and low running costs.Operation of a CE system involves application of a high voltage(typically 10-30 kV) across a narrow bore capillary (typically 25-100Fm). The capillary is filled with electrolyte solution which conductscurrent through the inside of the capillary. The ends of the capillaryare dipped into reservoirs filled with the electrolyte. Electrodes madeof an inert material such as platinum are also inserted into theelectrolyte reservoirs to complete the electrical circuit. A smallvolume of sample is injected into one end of the capillary. Thecapillary passes through a detector, usually a UV absorbance detector,at the opposite end of the capillary. Application of a voltage causesmovement of sample ions towards their appropriate electrode usuallypassing through the detector. Different sample ions arrive at adetection part of the capillary at different times. The sample may belabeled with a fluorescent marker so that when the sample passes througha beam of light at the detector, the fluorescent marker fluoresces andthe fluorescence is detected as an electric signal. The intensity of theelectric signal depends on the amount of fluorescent marker present inthe detection zone. The plot of detector response with time is thengenerated which is termed an electropherogram.

[0006] CE is a particularly preferred separation method, as it allowsthe use of high electric fields, due to the capillary tube efficientlydissipating the resulting heat produced by the electric field. As such,the separations achieved are much better than the more traditionalelectrophoretic systems. In addition, multiple capillary tubes may beclosely spaced together and used simultaneously to increase samplethroughput.

[0007] In traditional CE systems, analysis or detection of the separatedcomponents is performed while the sample is still located within thecapillary, and may be accomplished using photometric techniques such asadsorbance and fluorescence. Adsorbance and fluorescence is whereexcitation light is directed toward the capillary tube, and lightemitted from the sample (e.g., fluorescence) is measured by a detector,thereby providing information about the separated components. Therefore,in these systems, excitation light directed at the sample, as well aslight emitted from the sample, must be transmitted through thecapillary's walls. A drawback of this approach is that the fused silicacapillaries typically used in capillary electrophoresis are poor opticalelements and cause significant scattering of light. The problemassociated with light scattering is exacerbated by having multiplecapillaries disposed side-by-side, as scattered excitation light fromone capillary interferes with the detection of samples in neighboringcapillaries.

[0008] One approach to solving the problem of on-capillary detection hasbeen to detect a sample after the sample emerges from the capillary in adetection cell having superior optical characteristics, e.g., a flatquartz chamber. In this system, a sample is transported from the outletof a capillary to the detection cell by electrophoresis under theinfluence of the same voltage difference used to conduct theelectrophoretic separation. Examples of this type of system aredisclosed in U.S. Pat. No. 5,529,679, which is incorporated herein byreference.

[0009] A variation of the above system replaces the capillary tubes witha series of parallel channels formed in a plate or chip, where thechannels are in fluid communication with a detection cell in a mannersimilar to that described above. This type of system is known as amicro-channel array.

[0010] While addressing some of the abovementioned problems, thedetection cell type CE system has drawbacks of its own. For example,excitation energy, such as light from a laser, has the tendency toscatter, thereby diminishing the energy's intensity as it transmittedthrough the detection cell.

[0011] A partial cross-section of a prior art detection cell 102 isshown in FIG. 1A. The detection cell 102, typically made from glass,forms a cavity 108, which is filled with an electrolytic polymer 110containing a sample to be detected. Rays of light 104, typically from alaser, enter the detection cell 102 at a first end 112. Because thefirst end 112 is normal to the rays of light 104, the light 104 does notscatter, i.e., reflect or refract, when passing into the detection cell,from air to glass. However, when the light 104 passes through theboundary 106 between the detection cell and the polymer 110, the lightis refracted. This is due to the angle or slope of the boundary 106, andthe difference in refractive indices of the glass and polymer. The angleor slope of the boundary 106 is caused by current etching and masteringtechnologies, which are unable to produce optically flat vertical cavitywalls in glass or plastic.

[0012] The refracted light obeys the law of refraction, i.e.,

RI ₁ sin(A _(t))=RI _(R) sin(A _(R))

[0013] Where RI₁=first refractive index;

[0014] A₁=angle of incidence;

[0015] RI_(R)=second refractive index; and

[0016] A_(R)=angle of refraction.

[0017] As the polymer has a refractive index (approximately 1.41) lessthan the refractive index of glass (approximately 1.52), the angle ofrefraction is larger than the angle of incidence and the light bendsfurther away from the normal to the boundary 106. Many light rays arelost due to light escaping 116 out of the detection cell instead ofbeing trapped in the cavity by Fresnel reflection. This degrades theintensity of excitation light incident on the samples, which in turnadversely effects the detected signal strength. Furthermore, refractedlight rays may also reflect 114 off the internal surfaces of the cavity108 causing interference and detection signal loss. In other words, thecurved or angled interfaces or boundaries in combination with theunfavorable refractive index change at the glass to polymer boundary orinterface, leads to unsatisfactory light intensity and quality, leadingto poor sample detection.

[0018] Moreover, the optical channels of these systems are oftenextended along a narrow tunnel to a dead end at the light receiving sideof the detection cell. This introduces an interface, at the point wherethe tunnel meets the detection cell, that is hard to control, keepclean, and isolate from the high voltages in the detection cell.Further, the interface may cause a distortion of the electrophoresisfield. Still further, bubbles trapped in the tunnel may opticallyinterfere with the excitation light. This, in combination with poorlight intensity and quality, further aggravates the signal detection.

[0019] In light of the above drawbacks, there is a need for an improveddetection cell that provides better control over excitation light thatis used to detect or analyze separated sample components that have beenproduced using techniques such as CE tube or microchip technology.Further, there is a need for an improved method for controlling thedirection of the light rays within the detection cell.

SUMMARY OF THE INVENTION

[0020] According to the invention there is provided an energy beamguide. The energy beam guide comprises a first region having a firstrefractive index, the first region having an energy beam receiving endand an inclined first boundary opposing the energy beam receiving end.The energy beam guide also includes a second region having a secondrefractive index that is less than the first refractive index. Thesecond region shares the first boundary with the first region, and has adeclined second boundary opposing the first boundary. A predetermineddistance separates the first and second boundaries. Finally, the energybeam guide comprises a third region having a third refractive index. Thethird region shares the second boundary with the second region.

[0021] Further according to the invention there is provided anotherenergy beam guide. The energy beam guide comprises a first region havinga first refractive index and a second region sharing an inclined firstboundary with the first region. The second region has a secondrefractive index that is less than the first refractive index. Theenergy beam guide also includes a third region sharing a declined secondboundary with the second region. The third region has a third refractiveindex. Also, a predetermined distance separates the first and secondboundaries. The first refractive index is larger than the secondrefractive index, and preferably the second refractive index is largerthan the third refractive index.

[0022] Still further according to the invention there is provided adetection cell, preferably part of an electrophoresis system. Thedetection cell comprises a substrate and first and second cavitiesformed in the substrate. The first cavity has a first cavity sloped walland is configured to receive a first substance having a first refractiveindex. The substrate has a second refractive index. The second cavityhas a second cavity sloped wall and is configured to receive a secondsubstance having a third refractive index. A wall defined by a region ofthe substrate, separates the first and second cavities from each otherby a predetermined distance. The first refractive index is larger thanthe second refractive index, and preferably the second refractive indexis larger than the third refractive index.

[0023] According to the invention there is also provided a method formaking a detection cell. A substrate is firstly provided, where thesubstrate defines first and second cavities each having sloped walls andseparated by a wall. The first cavity is filled with a first substancehaving a first refractive index. The substrate is made from a substancehaving a second refractive index. The second cavity is then filled witha second substance having a third refractive index. The first refractiveindex is larger than the second refractive index, and preferably thesecond refractive index is larger than the third refractive index.

[0024] Still further according to the invention there is provided amethod for detecting component parts of a sample. A sample is firstlyseparated into its component parts by electrophoresis. An energy beam isdirected at a first region having a first refractive index. The energybeam is then redirected towards a second boundary, where the redirectingoccurs at an inclined first boundary separating the first region from asecond region. The second region has a second refractive index. Theenergy beam is subsequently guided towards a third region that includesthe component parts of the sample. The guiding occurs at a declinedsecond boundary separating the second region from the third region. Thecomponent parts are then struck with the energy beam and energy emittedfrom the component parts is detected. Again, the first refractive indexis larger than the second refractive index, and preferably the secondrefractive index is larger than the third refractive index. Theredirecting and guiding steps 6 comprise refracting the energy beam,such that at the boundaries an angle or refraction of the energy beam islarger than an angle of incidence.

[0025] The invention has a number of advantages over the prior art, forexample:

[0026] 1. The excitation energy beam is guided to a first boundary bytotal internal reflection.

[0027] 2. The distance between the cavities or regions, and the indicesof refraction can be adjusted to compensate for the detractivedispersion that occurs at an entry interface of the second cavity.

[0028] 3. The distance between the cavities or regions provides aoptical interface with the second cavity while isolating the opticalpath from the polymer, chemistry and a high voltage. The first cavityand its elements are not exposed to the high pressures required torefill the separation medium, i.e, the polymer in the second cavity.

[0029] 4. The invention eliminates tunnels, which are filled with theseparation medium and require special cleaning and refilling proceduresto assure a clean optical interface and satisfactory bubble removal.

[0030] 5. The second cavity is fabricated by the same process, and atthe same time, as the first cavity. This assures proper alignment andconsistent dimensions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] For a better understanding of the nature and objects of theinvention, reference should be made to the following detaileddescription, taken in conjunction with the accompanying drawings, inwhich:

[0032]FIG. 1A is a partial cross-section of a prior art detection cell;

[0033]FIG. 1B is a detection cell having a blunted channel;

[0034]FIG. 1C is a detection cell with two cavities;

[0035]FIG. 2A is a partial top view of a capillary electrophoresissystem according to an embodiment of the invention;

[0036]FIG. 2B is a cross-sectional side view of the capillaryelectrophoresis system shown in FIG. 2A, taken along line BB′;

[0037]FIG. 3 is a enlarged view of part of FIG. 2B;

[0038]FIG. 4A is a partial cross-sectional side view of a capillaryelectrophoresis system incorporating a different optical element,according to another embodiment of the invention;

[0039]FIG. 4B is a partial cross-sectional side view of a capillaryelectrophoresis system incorporating another optical element, accordingto yet another embodiment of the invention;

[0040]FIG. 4C is a partial cross-sectional side view of a capillaryelectrophoresis system incorporating an optical element and a supportblock, according to yet another embodiment of the invention;

[0041]FIG. 5A is a graph of “RI₁ vs DISTANCE and % INTENSITY” accordingto an embodiment of the invention;

[0042]FIG. 5B is another graph of “RI₁ vs DISTANCE and % INTENSITY”according to another embodiment of the invention;

[0043]FIG. 6A is a flow chart of a method for guiding an energy beam inan electrophoresis system, according to an embodiment of the invention;and

[0044]FIG. 6B is another flow chart of a method for guiding an energybeam in an electrophoresis system, according to another embodiment ofthe invention.

[0045] Like reference numerals refer to corresponding parts throughoutthe several views of the drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] To address the drawbacks of the prior art, a number of differentembodiments were tested. Ray tracing was carried out based on idealgeometrical and theoretical models of light propagation. To fullydescribe the success of the preferred embodiments of the invention, theunsuccessful models are briefly described below in relation to FIGS. 1Band 1C.

[0047]FIG. 1B shows a detection cell having a blunted channel 118 forattempting to address the above described drawbacks. Here, a bluntedchannel 118 is used to eliminate any interference created by internalreflection. Rays of light that might reflect off the internal surfacesof the cavity 108 are refracted at another boundary or interface 130through an angle of reflection less than the angle of incidence. Thisbends the light rays further away from the cavity, avoiding internalreflection. Although this embodiment lessens interference effectsassociated with diffraction due to less curvature at a boundary 130, theintensity of the excitation light in the cavity is greatly diminished.

[0048]FIG. 1C shows a substrate 124 with two cavities. The substrate 124is made from a material such as plastic or glass having a refractiveindex of approximately 1.52. The substrate 124 forms a first emptycavity 120 containing air with a refractive index of approximately 1.00.The substrate 124 also forms a second cavity 126 that is filled with apolymer having a refractive index of approximately 1.41. Obeying the lawof refraction, the angle of refraction at a first boundary 122 betweenthe air and the glass is less than the angle of incidence of the lightpassing through the boundary. This means that the light is refracteddownward, as shown. Those beams that pass through a second boundary 128between the glass and the polymer are also refracted downward, as theangle of refraction is greater than the angle of incidence. Again, thisembodiment lowers the intensity of the light in the second cavity 126,due to the fact that some light does not reach the second boundary 128.

[0049] Neither of the embodiments shown in FIG. 1B nor FIG. 1C werefound to sufficiently address the drawbacks of the prior art. In lightof the above, the embodiments of the invention described below weredeveloped. FIGS. 2 and 3 below show a system for controlling theexcitation light according to a preferred embodiment of the invention.

[0050]FIG. 2A is a partial top view of a CE system 200 according to anembodiment of the invention. The CE system 200 preferably comprises anexcitation source 202, a detection cell 210, a first cavity 214, and asecond cavity 218. The excitation source 202 is configured to emit anenergy beam 204 at the detection cell 210. In a preferred embodiment,the excitation source 202 is an argon gas laser that emits excitationlight. For convenience, the energy beam 204 will sometimes be referredto as the excitation light, although it should be appreciated that anysuitable energy may be used.

[0051] The detection cell 210 comprises of a substrate 216 that forms afirst cavity 214 and a second cavity 218. The first cavity 214 ispreferably filled or cast with a first substance that is chosen for itsease of handling, adhesive properties, and optical properties, such astransparency and refractive index, hereafter known as the firstrefractive index (RI₁). In a preferred embodiment the first cavity 214is filled with an optical adhesive or a fluid having the firstrefractive index, which has a refractive index of between approximately1.47 and 1.61. An inlet 224 is preferably provided for filling the firstcavity 214 with the first substance. The first cavity 214 is preferablyconsiderably wider than the energy beam 204 to ensure that the beam doesnot come into contact with the rounded edges 215 on the sides of thefirst cavity 214.

[0052] The substrate 216 is preferably made from a second substancechosen for its ease of manufacture, and optical properties, such astransparency and refractive index, hereafter known as the secondrefractive index (RI₂). The second substance is any material throughwhich light can propagate and that preferably has a refractive index inthe range of 1.46 to 1.52. In a preferred embodiment the secondsubstance is glass, which has a refractive index of approximately 1.52or 1.472. This refractive index was chosen because there are manysuitable plastics that have similar refractive indices and that may besubstituted for the glass to lower costs.

[0053] The second cavity 218 contains a third substance 223 chosen forits ability to conduct electricity, hold samples in suspension, and itsoptical properties, such as transparency and refractive index, hereafterknown as the third refractive index (RI₃). In a preferred embodiment,the third substance 223 is a polymer, which has a refractive index inthe range of 1.33 to 1.46. In a preferred embodiment, the thirdrefractive index is 1.41. In a preferred embodiment, the second cavity218 is where the detection of a sample entrained in the third substance223 is detected.

[0054] To achieve optimal intensity and quality of the excitationenergy, the overall relationship between the refractive indices ispreferably as follows:

RI₁>RI₂>RI₃

[0055] Capillaries or channels 212 open at their outlet ends into thesecond cavity 218 as described in the background section above. Anotherinlet 220 is preferably provided for filling the second cavity 218 withthe third substance 223.

[0056] An optical element 206 is preferably coupled to the substrate 216in the path of the energy beam 204. The optical element 206 ispreferably an optical flat, such as a flat glass disc having veryaccurately polished surfaces, and is used to control the input opticalsurface. In a preferred embodiment, the optical element 206 isstructurally bonded to the substrate 216 by an optical adhesive in thefirst cavity 214.

[0057] In one embodiment, the first cavity may be open on the energybeam receiving end of the substrate 216 nearest to the excitation source202. In this embodiment where there is no wall separating the opticalelement from the first cavity, the optical element is easily affixed tothe substrate 216. This is accomplished by placing the optical elementadjacent the first cavity 214, filling the first cavity with adhesive,and allowing some adhesive to flow between the optical element and thesubstrate 216. When the adhesive dries, the optical element will haveadhered to both the adhesive in the first cavity, and to the adhesive,which has leaked out of the first cavity 214 onto a sidewall of thesubstrate 216 bounding the first cavity 214.

[0058] In the embodiment where a liquid index matching fluid is used,instead of an adhesive, the optical element may be bonded to thesubstrate 216 prior to the filling of the first cavity, or it may beheld in place by an external support. Alternatively, no optical flat isnecessary where the second substance in the first cavity is cast with anoptically flat surface exposed to the excitation source.

[0059]FIG. 2B is a cross-sectional side view of the CE system shown inFIG. 2A, taken along line BB′. The side view clearly shows the first andsecond cavities 214 and 218, respectively. As described above, thecavities do not have rectilinear walls, but rather sloped or curvedboundaries 226 and 228, due to the limitations of etching technologiescurrently available for glass and plastic masters. In a preferredembodiment, the first boundary 226 has an inclined or concave shape,while the second boundary has a declined or convex shape, as presentedto the incident energy beam 204. This leads to a refraction of theenergy beam 204 at the boundaries 226 and 228, as explained in relationto FIG. 3 below.

[0060]FIG. 3 is an enlarged view of portion 230 of FIG. 2B. Toillustrate the light guiding principles of the invention, nine lightrays 312 are shown passing through the first cavity 214, first boundary226, substrate 216, second boundary 228, and second cavity 218,respectively. It should however be appreciated that any suitable energybeam may be used instead of the light rays. As described above, thefirst cavity 214 is filled with a first substance 222 having a firstrefractive index RI₁; the substrate 216 is made from a second substance304 having a second refractive index RI₂; and the second cavity 218 isfilled with a third substance 223 having a third refractive index RI₃.Also as described above, the substances 222, 304, and 223 are chosen sothat RI₁>RI₂>RI₃. In other words, the first substance 222 contained bythe first cavity 214 forms a first region, the third substance 223contained in the second cavity 218 forms a third region, and thesubstrate 216 made from the second substance 304 forms a second region,which separates the first and second regions by a distance 302.

[0061] Light rays 1 and 2 pass through the first and second boundary 226and a second boundary 228 normal to these boundaries. Therefore, littleor no refraction occurs for these rays. Boundaries 226 and 228 typicallyhave a curved shape, and, therefore, the angle of incidence of the lightrays incident on these boundaries increases as the angle of the inclinedslope increases. In other words, the angle of incidence of light ray 3striking first boundary 226 is smaller than the angle of incidence oflight ray 9 striking first boundary 226. Due to the orientation of theinclined slope of the first boundary 226, as well as the fact that RI₁is larger than RI₂, the angle of refraction will be larger than theangle of incidence at the first boundary 226, thereby bending the lightrays upward. Similarly, due to the orientation of the declined slope ofthe second boundary 228, as well as the fact that RI₂ is larger thanRI₃, the angle of refraction will also be larger than the angle ofincidence at the second boundary 228, thereby bending the light raysdownward. In other words, the incline or decline boundaries effectivelydetermine whether the light is bent upwards or downwards. The cumulativeeffect of both surfaces is to result in an exiting light ray that is asparallel to the channel surface 306 as possible. Furthermore, thedistance 302 between the first and second boundaries 226 and 228respectively, affects the path of the light rays, specifically how closethe rays of light remain to each other.

[0062] It should be noted that boundaries 226 and 228 are contiguous. Noother substance forms a layer between each region at the boundary, i.e,there is no air or other substance, such as adhesive, separating theregions from one another.

[0063] Furthermore, because RI₁ is preferably larger than RI₂, any lightin the first cavity 214 is reflected inside the channel by totalinternal reflection. Because RI₃ is preferably less than RI₂, anyreflection that occurs within the second cavity 218 is due to Fresnelreflection. Fresnel reflection is only 80% efficient where the angle ofincidence of incident light, is larger than 89 degrees, i.e., the beamangle is within 1 degrees of the reflecting surface.

[0064] Therefore, by selecting the refractive indices RI₁, RI₂, and RI₃,and the distance 302 between the boundaries 226 and 228, the energy beam(e.g. light rays) can be accurately guided through the detection cell.This retains the intensity of light in the second cavity 218 and reducesthe amount of internal reflection by reducing the amount of light lostto rays that exceed the Fresnel reflection angle, which is less than 2degrees for a 90% efficiency reflection. In a preferred embodiment, thedistance 302 is between 0.1 and 1000 microns.

[0065]FIG. 4A is a partial cross-sectional side view of a CE system 400incorporating a different optical element 402, according to anotherembodiment of the invention. The entry surface 404 of the opticalelement 402 is angled to facilitate an energy beam 204 that strikes theoptical element at an angle, and may improve fabrication by allowing fordraft, which is the angle allowed on component sides to facilitatecomponent removal from a mold, i.e., injection molded optics.

[0066]FIG. 4B is a partial cross-sectional side view of a CE system 406incorporating another optical element 408, according to yet anotherembodiment of the invention. The optical element 408 is preferably castfrom, and at the same time as, the first substance 222 (FIG. 2) is castinto the first cavity 214. The cast optical element 408 may be cast inany suitable shape, but is preferably cast as a cylindrical lens thatfocuses the energy beam 204 into the first cavity 214.

[0067]FIG. 4C is a partial cross-sectional side view of a capillaryelectrophoresis system incorporating an optical element and a supportblock, according to yet another embodiment of the invention. Thisembodiment allows for the first substance to be a liquid, such as anindex matching fluid. In this embodiment a support block 416 is used tosupport and align an optical element 412. The support block 414 forms alower floor of a first cavity holding a first substance from which thefirst region 414 is composed. The support block 414 is preferably madefrom a plastic, glass, or ceramic.

[0068] The portion of the system not shown in FIGS. 4A, 4B, and 4C isthe same as that shown and described in relation to FIGS. 2 and 3 above.

[0069]FIG. 5A is a graph 500 of “RI₁ vs DISTANCE and % INTENSITY”according to an embodiment of the invention. “DISTANCE” refers to thedistance 302 (FIG. 3) between the first boundary 226 (FIG. 3) and thesecond boundary 228 (FIG. 3) at their closest distance to one another,i.e, measured along the shortest possible segment separating them.“FIRST REFRACTIVE INDEX” refers to the first refractive index (RI₁),while “% INTENSITY” refers to the percentage intensity of the energybeam at approximately 17 mm into the second cavity (third region) 218(FIG. 3) from the second boundary, as measured in a direction away fromthe first and second regions. As can be seen, the % Intensity increasesby a few percentage points for larger first refractive indices (RI₁),while the gap can be made significantly smaller by increasing the firstrefractive index (RI₁). Therefore, a small gap with a high firstrefractive index (RI₁) is preferred. In this embodiment, the preferredsubstances 222, 304, and 223 (FIG. 3) are as follows: first substance222 (FIG. 3) is an adhesive made by ABLESTIK, MASTERBOND, or NYE OPTICALhaving a first refractive index in the range of 1.56 to 1.57 inclusive;second substance 304 (FIG. 3) is 0211 CORNING GLASS (made by CORNING)having a second refractive index of 1.52, or a ZEONOR 1020R plastichaving a second refractive index of 1.523; and third substance 223 (FIG.3) is APPLIED BIOSYSTEMS POP-6 POLYMER GEL MATRIX having a thirdrefractive index of 1.41. The refractive index of the optical element isboth unknown and unimportant.

[0070]FIG. 5B is another graph 502 of “RI₁ vs DISTANCE and % INTENSITY”according to another embodiment of the invention. This chart is similarto that shown in FIG. 5A. The chart shows that for an intensity above0%, the first refractive index (RI₁) must lie in the range of 1.47 to1.52 inclusive, a suitable intensity and distance occurring at an RI₁ ofabout 1.50, and a distance of 420 microns. The highest intensity occursat approximately 30 microns, but this distance is too small formanufacturing processing purposes. In this 13 embodiment, the preferredsubstances 222, 304, and 223 (FIG. 3) are as follows: first substance222 (FIG. 3) is CARGILLE LIQUID INDEX MATCH fluid (made by CARGILLELABORATORIES, Inc.) having a first refractive index of 1.50; secondsubstance 304 (FIG. 3) is BOROFLOAT GLASS (made by SCHOTT GLASS) havinga second refractive index of 1.472; and third substance 223 (FIG. 3) isPOP-6 POLYMER GEL MATRIX (made by APPLIED BIOSYSTEMS) having a thirdrefractive index of 1.41. The refractive index of the optical element isboth unknown and unimportant, although a suitable optical element may bea VWR 48393-070 (made by VWR Scientific Products).

[0071] One skilled in the art will appreciate that once the firstsubstance and the second substance are selected, selections of asuitable distance 302 (FIG. 3) and a suitable first refractive index(RI₁) may be made by utilizing graph 500.

[0072]FIG. 6A is a flow chart 600 of a method making an energy beamguide, according to an embodiment of the invention. First, second, andthird substances are initially selected (at 602), such that RI, islarger than RI₂, which is larger than RI₃. In a preferred embodiment,RI₃ is available in a narrow range of indices. RI₂ is selected as thesubstrate and RI, is determined based on RI₂ and RI₃ from charts,iterative calculations, from a model, or through experimentation. Thedistance or wall between the first and second cavities is then selected(at 604). A substrate is provided (at 605) and defines first and secondcavities each having sloped walls and separated by a wall. The substrateis made from the second substance which has a second refractive index(RI₂). The first cavity is filled (at 606) with a first substance havingthe first refractive index (RI₁). The second cavity is filled (at 608)with a second substance having the third refractive index (RI₃).

[0073]FIG. 6B is a flow chart 612 a method for detecting component partsof a sample, comprising. Initially, a sample is separated (at 614) intoits component parts by electrophoresis. An energy beam is then directed(at 616) at a first region having a first refractive index (RI₁). Theenergy beam is subsequently redirected (at 618) towards a secondboundary, where the redirecting occurs at an inclined first boundaryseparating the first region from a second region having a secondrefractive index. The energy beam is then guided (at 620) towards athird region that includes component parts of a sample. The guidingoccurs at a declined second boundary separating the second region fromthe third region. The component parts are subsequently struck (at 622)with the energy beam. Finally, energy emitted from the component partsis detected (at 624). The first refractive index (RI₁) is larger 14 thanthe second refractive index (RI₂), and the second refractive (RI₂) indexis larger than the third refractive index (RI₃).

[0074] In the above described embodiments, where RI₁>RI₂>RI₃, optimalillumination is provided by directing the energy beam as parallel to thecavity surface 306 (FIG. 3) as possible, thereby, reducing the amount ofreflections in the cavities. In an alternative embodiment, however, RI,>RI₂>RI₃. In this embodiment, where the second refractive index is lessthan the third refractive index, light entering the third cavity isinternally reflected by total internal reflection. This allows theembodiment to function through bouncing illumination. This embodiment,however, necessitates locating a suitable polymer with a refractiveindex greater than the substrate's refractive index.

[0075] The foregoing descriptions of specific embodiments of the presentinvention are presented for purposes of illustration and description.They are not intended to be exhaustive or to limit the invention to theprecise forms disclosed, obviously many modifications and variations arepossible in view of the above teachings. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical applications, to thereby enable others skilled in the artto best utilize the invention and various embodiments with variousmodifications as are suited to the particular use contemplated.Furthermore, the order of steps in the method are not necessarilyintended to occur in the sequence laid out. It is intended that thescope of the invention be defined by the following claims and theirequivalents.

What is claimed is:
 1. An energy beam guide, comprising: a first regionhaving a first refractive index, said first region having an energy beamreceiving end and an inclined first boundary opposing said energy beamreceiving end; a second region having a second refractive index that isless than said first refractive index, said second region sharing saidfirst boundary with said first region, and having a declined secondboundary opposing said first boundary, where a predetermined distanceseparates said first and second boundaries; and a third region having athird refractive index, said third region sharing said second boundarywith said second region.
 2. The energy beam guide of claim 1, whereinsaid second refractive index is larger than said third refractive index.3. The energy beam guide of claim 1, wherein said second refractiveindex is less than said third refractive index.
 4. The energy beam guideof claim 1, wherein said energy beam guide forms part of a detectioncell of an electrophoresis system.
 5. The energy beam guide of claim 4,wherein said third region defines a detection portion of said detectioncell.
 6. The energy beam guide of claim 1, further comprising anexcitation source and a detector.
 7. The energy beam guide of claim 1,wherein said first refractive index is in a range from 1.47 to 1.61. 8.The energy beam guide of claim 1, wherein said second refractive indexis in a range from 1.46 to 1.52
 9. The energy beam guide of claim 1,wherein said second refractive index is 1.52.
 10. The energy beam guideof claim 1, wherein said second refractive index is 1.472.
 11. Theenergy beam guide of claim 1, wherein said third refractive index is isin a range from 1.33 to 1.46
 12. The energy beam guide of claim 1,wherein said third refractive index is 1.41.
 13. The energy beam guideof claim 1, wherein said first region is an optical adhesive.
 14. Theenergy beam guide of claim 1, wherein said first region is a liquidindex matching fluid.
 15. The energy beam guide of claim 1, wherein saidsecond region is selected from a group consisting of glass and plastic.16. The energy beam guide of claim 1, wherein said third region is amigration medium.
 17. The energy beam guide of claim 16, wherein saidmigration medium is a polymer.
 18. The energy beam guide of claim 1,wherein said inclined first boundary presents a concave shape to saidenergy beam.
 19. The energy beam guide of claim 1, wherein said declinedsecond boundary presents a convex shape to said energy beam.
 20. Theenergy beam guide of claim 1, wherein said energy beam is refracted atsaid first and second boundaries.
 21. The energy beam guide of claim 20,wherein an angle of refraction is greater than the angle of incidence atboth said first and second boundaries.
 22. The energy beam guide ofclaim 1, wherein a shortest distance separating said first region fromsaid second region is in a range from 0.1 to 1000 microns.
 23. Theenergy beam guide of claim 1, further comprising an optical elementdisposed between an energy beam source and said energy beam guide. 24.The energy beam guide of claim 23, wherein an energy beam receiving endof said optical element is sloped.
 25. The energy beam guide of claim23, wherein said optical element is formed from a substance thatcomprises said first region.
 26. An energy beam guide, comprising: afirst region having a first refractive index; a second region sharing aninclined first boundary with said first region, said second regionhaving a second refractive index that is less than said first refractiveindex; and a third region sharing a declined second boundary with saidsecond region, said third region having a third refractive index, wherea predetermined distance separates said first and second boundaries. 27.The energy beam guide of claim 26, wherein said second refractive indexis larger than said third refractive index.
 28. The energy beam guide ofclaim 26, wherein said second refractive index is less than said thirdrefractive index.
 29. The energy beam guide of claim 26, wherein saidenergy beam guide forms part of a detection cell of an electrophoresissystem.
 30. The energy beam guide of claim 29, wherein said third regiondefines a detection portion of said detection cell.
 31. The energy beamguide of claim 26, further comprising an excitation source and adetector.
 32. The energy beam guide of claim 26, wherein said firstrefractive index is in a range from 1.47 to 1.61.
 33. The energy beamguide of claim 26, wherein said second refractive index is in a rangefrom 1.46 to 1.52
 34. The energy beam guide of claim 26, wherein saidsecond refractive index is 1.52.
 35. The energy beam guide of claim 26,wherein said second refractive index is 1.472.
 36. The energy beam guideof claim 26, wherein said third refractive index is is in a range from1.33 to 1.46
 37. The energy beam guide of claim 26, wherein said thirdrefractive index is 1.41.
 38. The energy beam guide of claim 26, whereinsaid first region is an optical adhesive.
 39. The energy beam guide ofclaim 26, wherein said first region is a liquid index matching fluid.40. The energy beam guide of claim 26, wherein said second region isselected from a group consisting of glass and plastic.
 41. The energybeam guide of claim 26, wherein said third region is a migration medium.42. The energy beam guide of claim 41, wherein said migration medium isa polymer.
 43. The energy beam guide of claim 26, wherein said inclinedfirst boundary presents a concave shape to an energy beam.
 44. Theenergy beam guide of claim 26, wherein said declined second boundarypresents a convex shape to an energy beam.
 45. The energy beam guide ofclaim 26, wherein an energy beam is refracted at said first and secondboundaries.
 46. The energy beam guide of claim 45, wherein an angle ofrefraction is greater than the angle of incidence at both said first andsecond boundaries.
 47. The energy beam guide of claim 26, wherein ashortest distance separating said first region from said second regionis in a range from 0.1 to 1000 microns.
 48. The energy beam guide ofclaim 26, further comprising an optical element disposed between anenergy beam source and said energy beam guide.
 49. The energy beam guideof claim 48, wherein an energy beam receiving end of said opticalelement is sloped.
 50. The energy beam guide of claim 48, wherein saidoptical element is formed from a substance that comprises said firstregion.
 51. A detection cell, comprising: a substrate; a first cavityformed in said substrate, said first cavity having a first cavity slopedwall and configured to receive a first substance having a firstrefractive index, wherein said substrate has a second refractive index;a second cavity formed in said substrate, said second cavity having asecond cavity sloped wall and configured to receive a second substancehaving a third refractive index; and a wall defined by a region of saidsubstrate separating said first and second cavities from each other by apredetermined distance, wherein said first refractive index is largerthan said second refractive index.
 52. The detection cell of claim 51,wherein said second refractive index is larger than said thirdrefractive index.
 53. The detection cell of claim 51, wherein saidsecond refractive index is less than said third refractive index. 54.The detection cell of claim 51, wherein said detection cell forms partof a detection cell of an electrophoresis system.
 55. The detection cellof claim 51, wherein said second cavity defines a detection portion ofsaid detection cell.
 56. The detection cell of claim 51, furthercomprising an excitation source and a detector.
 57. The detection cellof claim 51, wherein said first refractive index is in a range from 1.47to 1.61.
 58. The detection cell of claim 51, wherein said secondrefractive index is in a range from 1.46 to 1.52
 59. The detection cellof claim 51, wherein said second refractive index is 1.52.
 60. Thedetection cell of claim 51, wherein said second refractive index is1.472.
 61. The detection cell of claim 51, wherein said third refractiveindex is in a range from 1.33 to 1.46
 62. The detection cell of claim51, wherein said third refractive index is 1.41.
 63. The detection cellof claim 51, wherein said first substance is an optical adhesive. 64.The detection cell of claim 51, wherein said first substance is a liquidindex matching fluid.
 65. The detection cell of claim 51, wherein saidsubstrate is selected from a group consisting of glass and plastic. 66.The detection cell of claim 51, wherein said second substance is amigration medium.
 67. The detection cell of claim 66, wherein saidmigration medium is a polymer.
 68. The detection cell of claim 51,wherein at least part of said first cavity sloped wall presents aconcave shape to an energy beam.
 69. The detection cell of claim 51,wherein at least part of said second cavity sloped wall presents aconvex shape to an energy beam.
 70. The detection cell of claim 51,wherein an energy beam is refracted at said wall.
 71. The detection cellof claim 70, wherein an angle of refraction is greater than the angle ofincidence at said wall.
 72. The detection cell of claim 51, wherein ashortest distance separating said first cavity from said second cavityis in a range from 0.1 to 1000 microns.
 73. The detection cell of claim51, further comprising an optical element disposed between an energybeam source and said detection cell.
 74. The detection cell of claim 73,wherein an energy beam receiving end of said optical element is sloped.75. The detection cell of claim 74, wherein said optical element isformed from the first substance.
 76. A method for making a detectioncell, comprising: providing a substrate defining first and secondcavities each having sloped walls and separated by a wall; filling saidfirst cavity with a first substance having a first refractive index,where said substrate has a second refractive index; filling said secondcavity with a second substance having a third refractive index; whereinsaid first refractive index is larger than said second refractive index.77. The method of claim 76, comprising the initial step of selectingsaid first substance, said substrate, and said second substance, suchthat said first refractive index is larger than said second refractiveindex, and said second refractive index is larger than said thirdrefractive index.
 78. The method of claim 76, comprising the initialstep of selecting said first substance, said substrate, and said secondsubstance, such that said first refractive index is larger than saidsecond refractive index, and said second refractive index is less thansaid third refractive index.
 79. The method of claim 76, comprising theinitial step of selecting a distance between said first and secondcavities. 2
 80. A method for detecting component parts of a sample,comprising: directing an energy beam at a first region having a firstrefractive index; redirecting said energy beam towards a secondboundary, where said redirecting occurs at an inclined first boundaryseparating said first region from a second region having a secondrefractive index; guiding said energy beam towards a third region thatincludes component parts of a sample, where said guiding occurs at adeclined second boundary separating said second region from said thirdregion; striking said component parts with said energy beam; anddetecting energy emitted from said component parts; wherein said firstrefractive index is larger than said second refractive index.
 81. Themethod of claim 80, further comprising the initial step of separatingsaid sample into its component parts by electrophoresis.
 82. The methodof claim 80, wherein said redirecting and said guiding steps compriserefracting said energy beam, such that an angle or refraction of saidenergy beam is larger than an angle of incidence, at said boundaries.